Project Summary DNA-protein cross-links (DPCs) are formed when proteins become covalently bound to DNA form spontaneously as a result of normal cellular processes such as lipid peroxidation, histone demethylation, DNA replication, transcription, and DNA repair. DPCs can be induced by exposure to anti-tumor drugs, transition metals, UV light, and ?-radiation. DPCs interfere with many biological processes and are implicated in the accelerated aging and increased cancer incidence observed in Ruijs-Aalfs syndrome patients. The goal of this application is to map DPC lesions along the genome, investigate how human cells recognize and remove these exceedingly bulky DPC lesions, and to identify the mechanisms by which they cause mutagenicity and cell death. Our central hypothesis is that unrepaired DPCs compromise the efficiency and accuracy of DNA replication and contribute to the toxicity and mutagenicity induced by the agents listed above. Our research plan focuses on three aims. First, will use next generation sequencing in combination with affinity pull down and protein precipitation to identify specific genomic regions susceptible to spontaneous and xenobiotic- induced DPC formation in human cells. Second, we will elucidate the role of proteolytic processing in DPC repair. Affinity capture, unbiased searches, and candidate gene-based approaches twill be used to identify proteins required for proteolytic processing and repair of DPCs, determine how cells convert DPCs to smaller peptide lesions (DpCs), and identity critical DNA repair proteins required for DPC removal from the genome. Third, we will investigate the effects of DPCs and DpCs on DNA replication. Our in vitro studies using DNA Pol ? showed that efficiency and fidelity of translesion synthesis past peptide DpCs is strongly dependent on DNA sequence context. We will examine the effects of sequence context on bypass efficiency and mutagenicity in human cells. The structural basis for the context effects on the efficiency and fidelity of bypass will be studied by molecular modeling and NMR studies. We will use a newly developed assay that employs pigyBac transposition of DpC or DPC containing DNA to examine the effects these lesions have on chromosome replication. These studies will for the first time examine the biological outcomes of structurally defined chromosomal DPCs in human cells.